Lactase solution and dairy product using same

ABSTRACT

[Problem] To provide a lactase solution having excellent thermal stability. 
     [Solution] A lactase solution in which the ratio of a lactase fraction having a molecular weight of about 120 kDa measured by SDS polyacrylamide gel electrophoresis is 20% or more.

TECHNICAL FIELD

The present invention relates to a lactase solution, and milk and dairyproducts using the same.

BACKGROUND ART

Lactose intolerance is a condition exhibiting various symptoms such asabdominal pain and diarrhea caused by lactose present in a food productsuch as a dairy product due to congenital insufficiency of decomposinglactose. Lactose is a disaccharide composed of galactose and glucose. Inorder to address lactose intolerance, decomposing in advance the lactosecontained in milk or the like to galactose and glucose using the lactaseenzyme is carried out in the food manufacturing industry.

Lactase solutions that are used for decomposing lactose contained inmilk or the like is conventionally produced by culturing alactase-producing microorganism, extracting the lactase from the insideof the cells, removing culture-derived contaminants, and purifying thelactase, followed by addition of a stabilizer and then filtersterilization.

Patent Literature 1 (JP S60-18394 B) discloses an invention which isdirected to a method for producing lactase from a culture of a certainstrain of Kluyveromyces lactis. According to this method, afterautolysis of the yeast cells, the resulting crude enzyme solution ispassed through a DEAE-cellulose column, resulting in a separation intotwo active fractions (lactases A and B) by elution with a concentrationgradient of NaCl. Patent Literature 1 discloses that these two activefractions are little different in enzymatic properties includingthermostability, except that they have a slight difference in pHstability, and thus an enzyme preparation can include a mixture of theseactive fractions.

From results of the genetic analysis of lactase derived fromKluyveromyces lactis, the lactase is a polypeptide consisting of 1,025amino acids and its molecular weight is presumed to be 117,618(Non-Patent Literature 1).

It is further described in Patent Literature 1 that the lactasedescribed therein has an optimum temperature of 40 to 50° C. and isinactivated 45% after 10 minute at 50° C. and 100% after 10 minutes at55° C. at pH 7.0. However, it is not described therein that this enzymewas actually used to decompose the lactose contained in milk. Therefore,Patent Literature 1 describes nothing about the problem of the decreasein enzyme activity when lactase is added to a raw material milk, inparticular inactivation of the enzyme when it is subjected to heat loadat or above 40° C. in the milk.

CITATION LIST Patent Literature

-   Patent Literature 1: JP S60-18394 B-   Patent Literature 2: JP 2004-534527 A-   Patent Literature 3: JP 2009-517061 A

Non Patent Literature

-   Non-Patent Literature 1: Poch et al., Gene 1992 Sep. 1; 118(1):55-63

SUMMARY OF INVENTION Technical Problem

The present invention is aimed at providing a lactase solution that issuperior in thermal stability.

Solution to Problem

The present inventors have found that a lactase has high thermalstability by increasing, among lactase species, the ratio of a lactasefraction forming a band at about 120 kDa on SDS polyacrylamide gelelectrophoresis (SDS-PAGE), leading to the completion of the presentinvention.

Therefore, according to the present invention, there are provided:

[1] A lactase solution comprising a lactase fraction having a molecularweight of about 120 kDa measured by SDS polyacrylamide gelelectrophoresis in a ratio of 20% or more;

[2] The lactase solution according to [1], wherein the sum of the ratioof the 120-kDa lactase fraction and a ratio of a lactase fraction havinga molecular weight of about 80 kDa measured by SDS polyacrylamide gelelectrophoresis is 30% or more;

[3] The lactase solution according to [1], wherein a ratio of a lactasefraction having a molecular weight of about 50 kDa measured by SDSpolyacrylamide gel electrophoresis is 70% or less;

[4] The lactase solution according to any one of [1] to [3], wherein avalue obtained by dividing the sum of the ratio of the about 120-kDalactase fraction and the ratio of the about 80-kDa lactase fraction bythe ratio of the lactase fraction having the molecular weight of about50 kDa based on SDS polyacrylamide gel electrophoresis is 0.5 or more;

[5] The lactase solution according to any one of [1] to [4], for use inproducing dairy products;

[6] A dairy product containing the lactase solution according to any oneof [1] to [5];

[7] A method for treating a raw material milk, including adding to a rawmaterial milk the lactase solution according to any one of [1] to [5],and decomposing lactose contained in the raw material milk at 1 to 60°C.;

[8] A method for producing a lactase solution, including

a culture step in which a microorganism is cultured,

a collection step in which the lactase is collected from the cultureproduct obtained in the culture step, and

a purification step in which the lactase collected in the collectionstep is purified, wherein

the purification step includes one or more cycles of:

a step in which a salting-out treatment and a desalting treatment arecarried out;

[9] The method for producing the lactase solution according to [8],wherein the salting-out treatment includes

a saturation step in which to the collected lactase is added asalting-out agent to a degree of saturation of 10 to 90%, and

a keeping step in which after the saturation step, the lactase isallowed to stand at a temperature of 4 to 40° C. for a period of 1 to 80hours; and

[10] The method for producing the lactase solution according to [8] or[9], wherein the salting-out treatment is carried out at pH 4 to 9.

Advantageous Effects of Invention

According to the present invention, there is provided a lactase solutionin which the activity to decompose lactose is hardly reduced even whenit is added to a raw material milk and subjected to heat load at orabove 40° C.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of SDS-PAGE of various lactase solutions. Lane1: Example 1-1, lane 2: Example 1-2, lane 3: Comparative Example 1-1,lane 4: Comparative Example 1-2, lane 5: Comparative Example 2, lane 6:Comparative Example 3, and lane M: molecular weight standards. Lanes 1and 2, and lanes 3 and 4 show the results from different lots of lactasesolutions.

FIG. 2 shows the results of SDS-PAGE (panel A) and Western blotting(panel B) of lactose decomposition reactions in which lactase solutionswere added to raw material milk at 43° C. for 2 hours. In each of thepanels, lane 1: Example 1-1, lane 2: Example 1-2, lane 3: ComparativeExample 1-1, lane 4: Comparative Example 1-2, lane 5: ComparativeExample 2, lane 6: Comparative Example 3, and lane M: molecular weightstandards.

FIG. 3A represents graphs showing changes over time in the amount oflactose during the reaction in which a lactase solution was added to rawmaterial milk at 37, 40, 43, 46, and 49° C. to decompose lactosecontained therein. In the graphs, the results are plotted with □ forExample 1, ∘ for Comparative Example 1, ▴ for Comparative Example 2, andΔ for Comparative Example 3.

FIG. 3B represents graphs showing changes over time in the percentlactose decomposition during the reaction in which a lactase solutionwas added to raw material milk at 37, 40, 43, 46, and 49° C. todecompose lactose contained therein. In the graphs, the results areplotted with □ for Example 1, ∘ for Comparative Example 1, ▴ forComparative Example 2, and Δ for Comparative Example 3.

FIG. 4 shows the results of SDS-PAGE of lactase solutions havingdifferent ratios of the 120-kDa lactase fraction. Lane 1: Example 1-1,lane 2: Example 2, lane 3: Example 3, lane 4: Example 4, lane 5: Example5, lane 6: Comparative Example 1, and lane M: molecular weightstandards.

FIG. 5 represents graphs showing changes over time in the amount (panelA) and the percent decomposition (panel B) of lactose during thereaction in which a lactase solution having a different ratio of the120-kDa lactase fraction was added to raw material milks at 49° C. todecompose lactose contained therein. In the graphs, the results areplotted with □ for Example 1, ♦ for Example 2, ▴ for Example 3, x forExample 4, * for Example 5, and ∘ for Comparative Example 1.

DESCRIPTION OF EMBODIMENTS

The lactase that is used in the present invention is one of lactasesderived from yeasts (of the genus Kluyveromyces). Most of these lactasesare so-called neutral lactases having an optimum pH which is of frompH=6 to pH=8. Lactase producing yeasts of the genus Kluyveromyces are,for example, Kluyveromyces lactis, Kluyveromyces fragillis, andKluyveromyces marxianus.

A lactase solution of the present invention desirably has a lactaseactivity of 10 to 100,000 NLU/g. “NLU” stands for Neutral Lactase Unit.The method for measurement of lactase activity is as follows: The enzymeactivity of a lactase solution is determined by hydrolysis of asubstrate o-nitrophenyl-β-galactopyranoside (ONPG) into o-nitrophenoland galactose. The reaction is stopped by addition of sodium carbonate.Since the resulting o-nitrophenol is yellow in color in an alkalinemedium, changes in absorbance are used to determine the enzyme activity(expressed as NLU/g). This procedure is published in Food ChemicalsCodex (FCC), 4th ed., Jul. 1, 1996, pp. 801-802, Lactase (neutral)(β-galactosidase) activity.

A lactase solution of the present invention can be one including alactase that has been collected from a microorganism by the followingmethod and purified.

The method for producing a lactase solution of the present inventionundergoes four steps, that is,

(1) a step in which a microorganism is cultured,

(2) a step in which the lactase is collected from the microorganism,

(3) a step in which the lactase is purified, and

(4) a step in which the lactase activity of the lactase solution isadjusted.

The following gives a detailed explanation of these four steps.

Regarding (1) a step in which a microorganism is cultured, this step canbe performed employing a known medium and using a known microbialstrain. Culture conditions are also known, and can be selected as neededand as appropriate.

Regarding (2) a step in which the lactase is collected from themicroorganism, it is necessary that this step includes a step ofextracting the lactase from the microorganism when it is anintracellular enzyme. The extraction step is not limited in particular,as long as a method is used, which is capable of transferring thelactase to the outside of the cell, and known extraction methods can beused. On the other hand, if the lactase is an enzyme that is secretedoutside the cell of a microorganism modified by gene transfer,mutagenesis, or the like, then its cultured medium will contain thelactase, and thus the extraction step is not required.

Regarding (3) a step in which the lactase is purified, this step isimportant in order to obtain a lactase solution of the presentinvention. Patent Literatures 1, 2, and 3 are common in that each ofthese literatures uses chromatographic procedures to purify a lactasesolution. These chromatographic procedures advance the purification ofthe lactase, making it possible that the lactase solution has anincreased lactase activity. However, as described below, whenchromatographic procedures, such as partition or molecular sievechromatography, adsorption chromatography, or ion-exchangechromatography, were used to purify lactase, it turned out that theoriginal lactase of 120 kDa became decomposed into two lactase speciesof 80 kDa and 50 kDa. Both the lactase species having such molecularweights exhibit lactase activity; however, decomposition of the originallactase leads especially to an increase in the ratio of a 50-kDa lactasefraction, reducing the thermal stability of the lactase and as a result,giving rise to a problem that it becomes difficult that thedecomposition of lactose proceeds at relatively high temperatures.

The lactase of the present invention can be obtained by using saltingout and desalting treatments in the purification step. For example, thelactase of the present invention is obtained by precipitating thelactase by salting out, followed by collecting and re-dissolving theprecipitate, and then desalting the lactase to remove the saltscontained therein. Salting out of the lactase, and collecting andre-dissolution of and desalting of the precipitate can be carried out ina continuous manner. It is also possible to use in combination otherpurification procedures, including chromatography and active carbontreatments, as long as the lactase of the present invention is obtained.

The reason why the 120-kDa lactase species does not become decomposed bysalting out is assumed to be that salting out allows the lactase toprecipitate as macromolecules and to become insoluble, and thus thelactase has a decreased reactivity to decomposition.

Salting-out agents for salting out lactase include ammonium sulfate,sodium sulfate, potassium phosphate, magnesium sulfate, sodium citrate,sodium chloride, and potassium chloride. These agents can be used alonein combination of two or more.

When ammonium sulfate as a salting-out agent is added to a lactasecontaining solution, ammonium sulfate is added thereto, preferably to adegree of saturation of 10 to 90%, further preferably 30 to 70%. Incases where a different salting-out agent is used, it can be added in anamount which corresponds to that of ammonium sulfate added.

The lactase can be precipitated out of a lactase containing solution byaddition of a salting-out agent such as ammonium sulfate. It ispreferable that from the addition of a salting-out agent to when thelactase has been precipitated, the lactase containing solution isallowed to stand at a temperature of 1 to 40° C. for a period of 1 to 80hours. For pH conditions during this period, a pH of 4 to 9 ispreferable. Further preferably, the lactase containing solution isallowed to stand at a temperature of 4 to 25° C. (room temperature) fora period of 1 to 48 hours at a pH of 5 to 8. The lower limit oftemperature conditions can be set to a temperature at which the lactasecontaining solution does not become solidified. The liquid from whichthe lactase has been precipitated and the lactase containing precipitateare solid-liquid separated by filtration, and then the lactase in theform of solid is dissolved in water, buffer, or the like, and desaltedby dialysis or by ultrafiltration concentration.

Regarding (4) a step in which the lactase activity of the lactasesolution is adjusted, there is no limitation, as long as the lactaseactivity of the lactase solution can be adjusted. For example, this stepincludes addition of water, addition of aqueous solutions containingsalts, addition of stabilizer, and the like.

A lactase solution of the present invention can also be obtained bymixing a commercially available lactase solution and a lactase solutionobtained by the above-described method, as long as the ratios of lactasefractions with specified molecular weights measured by SDSpolyacrylamide gel electrophoresis meet to be within particular ratios.

The molecular weight of lactase species in a lactase solution can beroughly determined by SDS-PAGE using a 10% polyacrylamide gel. Forexample, a sample of a lactase solution is diluted in purified water, ifnecessary, and mixed 1:1 with an SDS-PAGE sample buffer, and the mixtureis heated at 95 degrees for 5 minutes to prepare an electrophoresissample. A 10% acrylamide gel is loaded with standards and the preparedelectrophoresis sample, and subjected to electrophoresis. The standardsuse, for example, BIO-RAD #161-0313 (pre-stained) standards. The gelafter electrophoresis is subjected to protein staining with a CBBstaining solution (APRO SP-4010).

After a lactase solution of the present invention is subjected toSDS-PAGE and CBB staining, the gel is dried using the TEFCOpolyacrylamide drying kit (a trade name of Clear Dry Solution). Thedried gel is scanned as a gray-scale image with an EPSON scannerGT-X820, on which the densities of stained protein bands (correspondingto protein amounts) are determined using the Image J software (NIH,Bethesda, Md.).

A lactase solution of the present invention has a high ratio of alactase fraction having a molecular weight of about 120 kDa whendetermined by the above-described method. In the present invention, a“lactase fraction having a molecular weight of about 120 kDa measured bySDS polyacrylamide gel electrophoresis” refers to a fraction of alactase species forming a band located at a position corresponding toabout 120 kDa (or in a range between about 100 kDa and about 150 kDa),relative to the mobilities of molecular weight standards, afterelectrophoresis is performed using the above-described method.

For a lactase solution of the present invention, the ratio of an about120-kDa lactase fraction determined by the Image J software (NIH,Bethesda, Md.) after SDS-PAGE and CBB staining is 20% or more,preferably 50% or more, further preferably 80% or more, and mostpreferably 90% or more. Its upper limit is not particularly limited,but, for example, 100%. The ratio of such a lactase fraction iscalculated by the method described below.

After SDS-PAGE and CBB staining, the Image J software (NIH, Bethesda,Md.) is used to quantify the densities of stained protein bands tocalculate the ratio of a stained protein bands corresponding to about120 kDa on the basis of the total main bands as 100%, including stainedprotein bands corresponding to about 120 kDa (100 to 150 kDa), about 80kDa (80 to 100 kDa), about 50 kDa (49 to 54 kDa), and about 30 kDa (28to 32 kDa). The values recited in claims of the present application arevalues calculated when the total of these four stained protein bands isset to be 100% unless specifically recited.

For a lactase solution of the present invention, the sum of the ratio ofan about 80-kDa lactase fraction calculated using the above-describedmethod and the ratio of the above-described 120-kDa lactase fraction,preferably is 30% or more, more preferably 60% or more, furtherpreferably 90% or more. Its upper limit is not particularly limited, butfor example, 100%.

The decomposition of a lactase species of about 120 kDa (which may bereferred to hereinafter as lactase I) generates a lactase species ofabout 80 kDa (which may be referred to hereinafter as lactase II), andthe decomposition of lactase I or II generates a lactase species ofabout 50 kDa (which may be referred to hereinafter as lactase III).

Lactases I and II in a lactase solution each have lactase activity andheat resistance. Lactase III has lactase activity, but is poor in heatresistance. Any of these lactase fractions has comparable lactaseactivity.

Since lactase II is a decomposition product of lactase I, it ispreferable that the ratio of lactase I in a lactase solution is largerthan that of lactase II from a viewpoint of heat resistance.

The ratio of lactase III in a lactase solution preferably is 70% orless, more preferably 40% or less, further preferably 10% or less. Itslower limit is not particularly limited, but for example 0%.

The value obtained by dividing the sum of the ratios of lactases I andII by the ratios of lactase III preferably is 0.5 or more, morepreferably 1.0 or more, further preferably 5.0 or more. When the valueis less than 0.5, there is a tendency that the lactase solution exhibitsinsufficient heat resistance. Its upper limit is not limited because itis most preferable that the lactase solution contains no lactase III,that is, zero amounts of lactase III.

A raw material milk is a subject to which the lactase solution is to beadded. In the present invention, known raw material milks can beemployed. Raw material milks also include ones before and afterpasteurization. Any raw material milk can be used, as long as it isobtained using a milk. Ingredients composing raw material milks arewater, raw milk, pasteurized milk, defatted milk, dry whole milk,non-fat dry milk, buttermilk, butter, cream, whey protein concentrates(WPCs), whey protein isolates (WPIs), α (alpha)-La, β (beta)-Lg, and thelike.

The lactose contained in a raw material milk can be decomposed by addingthereto a lactase solution of the present invention. The decompositiontemperature is 1 to 60° C. and the decomposition time is 10 minutes to24 hours.

Examples of specific usages of a lactase solution include, for example,use in the production of a fermented milk. Methods for producing alactose-decomposed fermented milk include: for example, 1. a method inwhich a lactase solution is added to a milk before pasteurization,thereby to decompose lactose, followed by heat pasteurization of themilk and concomitant inactivation of the lactase, followed byfermentation of the treated milk (JP Hei 5-501197 A); 2. a method inwhich a lactase solution is added to a pasteurized milk, thereby todecompose lactose, followed by heat treatment to inactivate the lactase,followed by fermentation of the treated milk; 3. a method in which thelactose in a milk is decomposed by an immobilized lactase, followed byfermentation of the treated milk (JP S46-105593 A and S59-162833 A); and4. a method in which a raw material that has been subjected beforehandto decomposition or removal of lactose is used as a pasteurized milk,followed by fermentation.

A lactase solution according to the present invention is particularlysuitable for use in the production of dairy products. Here, dairyproducts refer to ice cream, milks such as long life milk, yogurt, freshcream, sour cream, cheese, and others. Particularly, a lactase solutionaccording to the present invention can preferably be used when exposedto heat load at or above 40° C. Such use includes, for example, use inthe production of yogurts.

EXAMPLES 1. Production of Lactase Solutions Example 1

A liquid medium containing 7% corn steep liquor and 2% lactose waspressure-sterilized (and had a pH of 5.5 after sterilization), and wasinoculated with Kluyveromyces lactis No. 013-2 (strain ATCC 8585), whichwas cultured at 30° C. for 24 hours under aeration at 12000 L/min. Aftercompletion of the culturing, the culture broth was left to stand for 4hours with cooling. Then, the supernatant was removed from the top ofthe fermentation tank to obtain 1500 kg of cells, which had aggregatedand settled down at the bottom of the tank. Then, 1500 g of thusobtained cells were washed with tap water. After that, 80 ml toluene wasadded to and mixed with the cells, followed by addition of 1500 ml of0.05 M phosphate buffer (pH 7.0). The mixture was stirred to make ahomogeneous suspension, which then was left to stand, under hermeticsealing, at 30° C. for 15 hours for cell autolysis.

The resulting autolysis product was centrifuged to obtain 2500 mlsupernatant, to which an equal volume of cold acetone was added, and themixture was left to stand overnight. The resulting precipitates werecollected by centrifugation, and then dissolved in 600 ml tap water toprepare an enzyme solution before concentration.

To the enzyme solution (600 ml) before concentration, with cooling to 4°C., was added ammonium sulfate powder in portions over 60 minutes tomake an aqueous solution that was 50% saturated therewith. The aqueoussolution was left to stand (left standing) at 4° C. for 80 hours toallow the lactase to settle down, followed by solid-liquid separation byfiltration to collect the lactase in the form of solid. The lactase wasre-dissolved in 600 ml tap water, followed by ultrafiltrationconcentration. To the resulting desalted lactase solution was added tapwater, followed by addition of glycerin to a final concentration of 50%to prepare a lactase solution (5,000 NLU/g) of Example 1.

Example 2

The lactase solution of Example 1 and a lactase solution of ComparativeExample 1 described below were mixed at a ratio by weight of 80:20 toprepare a lactase solution (5,000 NLU/g) of Example 2.

Example 3

The lactase solution of Example 1 and a lactase solution of ComparativeExample 1 described below were mixed at a ratio by weight of 60:40 toprepare a lactase solution (5,000 NLU/g) of Example 3.

Example 4

The lactase solution of Example 1 and a lactase solution of ComparativeExample 1 described below were mixed at a ratio by weight of 40:60 toprepare a lactase solution (5,000 NLU/g) of Example 4.

Example 5

The lactase solution of Example 1 and a lactase solution of ComparativeExample 1 described below were mixed at a ratio by weight of 20:80 toprepare a lactase solution (5,000 NLU/g) of Example 5.

Comparative Example 1

A commercially available lactase preparation, which is available as atrade name of “GODO-YNL 2SS” (manufactured by GODO SHUSEI CO., LTD.;5000 NLU/g), was used to prepare a lactase solution of ComparativeExample 1.

Comparative Example 2

A commercially available lactase preparation, which is available as atrade name of “MAXILACT LG 5000” (manufactured by DSM; 5000 NLU/g), wasused to prepare a lactase solution of Comparative Example 2.

Comparative Example 3

A commercially available lactase preparation, which is available as atrade name of “MAXILACT LGX 5000” (manufactured by DSM; 5000 NLU/g), wasused to prepare a lactase solution of Comparative Example 3.

2. Electrophoresis of Lactase Solutions

Each of these lactase solutions was diluted in milli-Q water to have alactase activity of 10 NLU/g, and mixed 1:1 with an SDS-PAGE samplebuffer (0.125 M Tris-HCl, pH 6.8, 0.0125% bromothymol blue, 20%glycerin, 2.5% SDS, 2.5% 2-mercaptoethanol), and the mixture was heatedat 95° C. for 5 minutes to prepare an electrophoresis sample. A 10%acrylamide gel (4% stacking gel, a thickness of 1 mm, an electrophoresisdistance of 50 mm) was loaded with molecular weight standards and theprepared samples and subjected to electrophoresis in a Marisol Industryelectrophoresis apparatus, which was run at a constant current of 10 mAin the stacking gel and 20 mA in the separating gel until the runningfront reached almost the lower end of the gel. The molecular weightstandards (lane M) used BIO-RAD #161-0313 (pre-stained) standards. Thegel after electrophoresis was subjected to protein staining for 1 hourwith a CBB staining solution (APRO SP-4010).

The results are shown in FIG. 1. For the lactase solution of Example 1(lanes 1 and 2), only a main band with a molecular weight of about 120kDa was observed. In contrast, for the lactase solution of ComparativeExample 1 (lanes 3 and 4), a band with a molecular weight of about 120kDa almost disappeared, while three bands with molecular weights ofabout 80 kDa, about 50 kDa, and about 30 kDa were detected as mainbands. For the lactase solutions of both Comparative Examples 2 (lane 5)and 3 (lane 6), in addition to a band with a molecular weight of about120 kDa, a band with a molecular weight of about 80 kDa was almost notobserved, and there were observed main bands at about 50 kDa and about30 kDa.

Lactase activity was detected for bands with molecular weights of about120 kDa, about 80 kDa, and about 50 kDa, and not for a band with amolecular weight of about 30 kDa.

3. Quantitative Determination of Each of the Lactase Bands and Results 1

The ratios of the respective lactase bands on the gel afterelectrophoresis were calculated based on their quantitativedetermination using the above-described method. The results are shown inTable 1. Table 1 lists calculated values for four lactase main bands onthe basis of all the protein bands. Furthermore, Table 2 shows theresults of the calculated ratios of the respective lactase bands on thebasis of only four lactase main bands in total. In Tables 1 to 4, thereason why the sum of the values for the lactase solutions of theExamples and Comparative Examples is not 100 is that their respectivemeasurements obtained were rounded off to one decimal place.

Although not shown in Tables 1 and 2, when fractions A and B obtained byrepeating Example 1 of Patent Literature 1 were analyzed byelectrophoresis as with Example 1 and others of the present invention,these fractions had a similar tendency to those observed for ComparativeExamples 2 and 3 of the present invention.

TABLE 1 Compar- Compar- Compar- Compar- Ex- Ex- ative ative ative ativeample ample Example Example Example Example 1-1 1-2 1-1 1-2 2 3 About120,000 78.8 76.6 13.0 8.7 3.9 3.5 About 80,000 23.9 14.1 5.3 3.5 About50,000 25.7 32.6 36.7 37.9 About 30,000 37.5 43.0 53.7 55.1 Others 21.223.5 1.6 0.4

TABLE 2 Compar- Compar- Compar- Compar- Ex- Ex- ative ative ative ativeample ample Example Example Example Example 1-1 1-2 1-1 1-2 2 3 About120,000 100.0 100.0 13.0 8.9 3.9 3.5 About 80,000 23.9 14.3 5.3 3.5About 50,000 25.7 33.1 36.9 37.9 About 30,000 37.5 43.7 53.9 55.1

4. Electrophoresis and Western Blot Analyses of Milks after LactoseDecomposition Reaction

To a milk that had gone through UHT pasteurization (at 130° C. for 2seconds) (a trade name of “Meiji Oishii Gyunyu”, manufactured by MeijiCo., Ltd.) was added the lactase solution of Example 1 (corresponding tolanes 1 and 2), or Comparative Example 1 (corresponding to lanes 3 and4), 2 (corresponding to lane 5), or 3 (corresponding to lane 6) to afinal concentration of 0.05% (w/v), and the mixture was subjected to alactose decomposition reaction at 43° C. for 2 hours. The solution afterthe reaction was diluted 20 times (w/v) in purified water, and mixed 1:1with an SDS-PAGE sample buffer, and the mixture was heated at 95 degreesfor 5 minutes to prepare an electrophoresis sample. Two 10% acrylamidegels were loaded with molecular weight standards and the preparedelectrophoresis samples, and subjected to electrophoresis in parallel.The molecular weight standards (lane M) used BIO-RAD #161-0313(pre-stained) standards.

After the electrophoresis, one of the gels was subjected to proteinstaining with a CBB staining solution as described above, and the otherto western blotting. As the western blotting transfer membrane, anitrocellulose membrane (BIO-RAD #162-0114) was used, and westernblotting was carried out in a wet manner. The membrane after thetransfer was blocked with a Block Ace solution (4 g Block Ace powder(Snow Brand Milk Products Co., Ltd.) in 100 ml purified water), and thenwashed with Tween-PBS.

As the primary antibody, an anti-lactase polyclonal antibody was used,which had been prepared in-house as described below. SDS-PAGE wasperformed using an aliquot of the lactase solution obtained in Example1, and a portion containing a 120-kDa lactase band was cut out of thegel and crushed, and then mixed with Difco Adjuvant, Complete, Freund,to make an emulsion. The emulsion was subcutaneously injected a total ofthree times at the base of the tail of Balb-C mice. After an increase inantibody titer in serum was observed, the supernatant obtained bycentrifugation of a collected whole blood sample was used as ananti-lactase polyclonal antibody. This antibody was capable of detectinglactase bands with molecular weights of 120, 80, and 50 kDa.

The membrane was subjected to reaction at room temperature for 2 hoursin a solution in which the anti-lactase antibody was diluted 1,000 timesin a diluted Block Ace solution, which was a 10-times dilution of theBlock Ace solution in purified water. The membrane was washed four timeswith Tween-PBS and then subjected to reaction at room temperature for 2hours in a solution in which a secondary antibody (gort a-mouseIgG(H+L)-HRP; SouthernBiotech, 1034-05) was diluted 5,000 times in adiluted Block Ace solution. The membrane was washed with Tween-PBS,followed by staining with 3,3′-diaminobenzidine (DAB). For the DABsubstrate, a DAB buffer tablet (MERCK, 1.02924.0001) was used asindicated.

The results are shown in FIG. 2. In the CBB staining image (panel A),there were observed almost the same results regardless of thedifferences of lactase solutions used. In contrast, the results ofwestern blotting (panel B) showed different bands due to the differencesof lactase solutions used, as in the case of the lactase solutionsbefore the reaction. That is, for the lactase solution of Example 1,only a main band with a molecular weight of about 120 kDa was observed;for the lactase solution of Comparative Example 1, a band with amolecular weight of about 120 kDa almost disappeared, while two bandswith molecular weights of about 80 kDa and about 50 kDa were detected asmain bands. For the lactase solution of Comparative Example 2 or 3, inaddition to a band with a molecular weight of about 120 kDa, a band witha molecular weight of about 80 kDa was almost not observed, and the mainband was observed at about 50 kDa.

Based on the above, there were not found changes in the molecularweights of and in the ratios of molecular weight fractions of thelactase species contained in each of the lactase solutions betweenbefore and after subjected to the lactose decomposition reaction.

5. Lactose Decomposition Test 1

To a milk that had gone through UHT pasteurization (at 130° C. for 2seconds) was added each of the lactase solutions of Example 1 andComparative Examples 1, 2, and 3 to a final concentration of 0.05% (w/v)(2.5 NLU per 100 mL milk), and the mixture was subjected to a lactosedecomposition reaction at 49° C., 46° C., 43° C., 40° C., and 37° C. Thecontents of lactose in the reaction solutions before and over timeduring the lactose decomposition reaction were determined by HPLC (usinga Transgenomic CARBOSep CHO620 column in the Waters Alliance HPLCsystem; column temperature: 85° C., solvent: H₂O, flow rate: 0.5 mL/min,detector: Waters 2414 RI detector). Percent lactose decomposition wascalculated according to the following formula: Percent lactosedecomposition (%)=100−[(the content of lactose in the milk after it wassubjected to the lactose decomposition reaction using the lactasesolution of an Example or Comparative Example)/(the content of lactosein the milk before it was subjected to the lactose decompositionreaction using the lactase solution of the Example or ComparativeExample)×100].

The results are shown in FIG. 3. FIGS. 3A and 3B show the results ofchanges over time in lactose content and in percent lactosedecomposition, respectively. After 2 hours of the lactose decompositionreaction at 37° C., no differences in percent lactose decomposition wereobserved for the lactase solution of Example 1 or Comparative Example 1,2, or 3, but there was found a tendency that, as the reactiontemperature was increased, the lactase solution of Example 1 gave thehighest percent of decomposition of lactose, followed by ComparativeExample 1, and the lactase solution of Comparative Example 2 or 3 gavethe lowest percent of decomposition of lactose. As has been mentioned,it was shown that also when the reaction temperature was increased, thelactase solution of the present invention exhibited no inactivation oflactase and thus a higher thermal stability. The lactase solution ofExample 1 also allows a more efficient decomposition of lactose in ashorter reaction time by increasing the reaction temperature. Also forlactase solutions from different lots according to the presentinvention, the reproducibility of these findings was verified.

Since the molecular weights of the main lactase fractions in therespective lactase solutions did not change between before and after thelactose decomposition reaction, the decrease in percent lactosedecomposition is attributable to the decrease in lactase activity duringthe reaction. It was also found that the higher the content of the120-kDa lactase fraction, the higher the percent lactose decompositionin the reactions at or above 40° C.

6. Examinations Using Mixed Lactase Solutions

Examinations were made of the lactase solutions of Examples 2 to 5,which were lactase solutions obtained by mixing the lactase solutions ofExample 1 and Comparative Example 1 at the above-described ratios.

6.1 Quantitative Determination of Lactase Bands and Results 2

For the lactase solutions of Examples 2 to 5, electrophoresis wasperformed by the above-described method, followed by quantitativedetermination of lactase bands. The results of electrophoresis of thelactase solutions are shown in FIG. 4 and the results of quantitativedetermination of lactase bands are shown in Table 3. Table 3 listsvalues for four main lactase bands when all the protein bands were usedfor calculation. Furthermore, Table 4 shows the results when only thefour main lactase bands were used to calculate the ratios of therespective lactase bands. For comparison, Tables 3 and 4 include theresults of electrophoresis and quantitative determination that werecarried out concurrently for the lactase solutions of Example 1 andComparative example 1.

TABLE 3 Ex- Ex- Ex- Ex- Ex- Comparative ample ample ample ample ampleExample 1 2 3 4 5 1 About 120,000 82.8 77.8 66.5 44.3 23.8 5.5 About80,000 1.9 2.2 6.3 7.7 12.9 About 50,000 11.7 16.3 26.3 32.7 38.8 About30,000 4.9 13.8 21.9 32.5 40.8 Others 17.2 3.7 1.2 1.1 3.4 2.0

TABLE 4 Ex- Ex- Ex- Ex- Ex- Comparative ample ample ample ample ampleExample 1 2 3 4 5 1 About 120,000 100.0 80.8 67.3 44.8 24.6 5.7 About80,000 2.0 2.2 6.4 7.9 13.1 About 50,000 12.1 16.5 26.6 33.8 39.6 About30,000 5.1 14.0 22.2 33.6 41.7

Lactose Decomposition Test 2

The lactase solutions of Examples 1 to 5 and Comparative Example 1 wereused for a lactose decomposition reaction at 49° C. by theabove-described lactose decomposition test. The results obtained areshown in FIG. 5.

Panels A and B show the results of changes over time in lactose contentand in percent lactose decomposition, respectively. It was found thatthe decomposition of lactose increased with an increasing ratio of theabout 120-kDa lactase band.

Therefore, it can be said from the above results that a lactase solutionin which the ratio of the main lactase band at about 120 kDa on SDS-PAGEis 20% or more has a higher heat resistance than that giving any of themain lactase bands with molecular weights of about 80 kDa, about 50 kDa,and about 30 kDa on SDS-PAGE.

1. A lactase solution comprising a lactase fraction having a molecularweight of about 120 kDa measured by SDS polyacrylamide gelelectrophoresis in a ratio of 20% or more.
 2. The lactase solutionaccording to claim 1, wherein the sum of the ratio of the about 120-kDalactase fraction and a ratio of a lactase fraction having a molecularweight of about 80 kDa measured by SDS polyacrylamide gelelectrophoresis is 30% or more.
 3. The lactase solution according toclaim 1, wherein a ratio of a lactase fraction having a molecular weightof about 50 kDa measured by SDS polyacrylamide gel electrophoresis is70% or less.
 4. The lactase solution according to claim 1, wherein avalue obtained by dividing the sum of the ratio of the about 120-kDalactase fraction and the ratio of the about 80-kDa lactase fraction bythe ratio of the lactase fraction having the molecular weight of about50 kDa measured by SDS polyacrylamide gel electrophoresis is 0.5 ormore.
 5. The lactase solution according to claim 1, for use in producingdairy products.
 6. A dairy product comprising the lactase solutionaccording to claim
 1. 7. A method for treating a raw material milk,comprising adding to a raw material milk the lactase solution accordingto claim 1, and decomposing lactose contained in the raw material milkat 1 to 60° C.
 8. A method for producing a lactase solution, comprisinga culture step in which a microorganism is cultured, a collection stepin which the lactase is collected from the culture product obtained inthe culture step, and a purification step in which the lactase collectedin the collection step is purified, wherein the purification stepincludes one or more cycles of: a step in which a salting-out treatmentand a desalting treatment are carried out.
 9. The method for producingthe lactase solution according to claim 8, wherein the salting-outtreatment comprises; a saturation step in which to the collected lactaseis added a salting-out agent to a degree of saturation of 10 to 90%; anda keeping step in which after the saturation step, the lactase isallowed to stand at a temperature of 4 to 40° C. for a period of 1 to 80hours.
 10. The method for producing the lactase solution according toclaim 8, wherein the salting-out treatment is carried out at pH 4 to 9.11. The lactase solution according to claim 2, for use in producingdairy products.
 12. The lactase solution according to claim 3, for usein producing dairy products.
 13. The lactase solution according to claim4, for use in producing dairy products.
 14. A dairy product comprisingthe lactase solution according to claim
 2. 15. A dairy productcomprising the lactase solution according to claim
 3. 16. A dairyproduct comprising the lactase solution according to claim
 4. 17. Amethod for treating a raw material milk, comprising adding to a rawmaterial milk the lactase solution according to claim 2, and decomposinglactose contained in the raw material milk at 1 to 60° C.
 18. A methodfor treating a raw material milk, comprising adding to a raw materialmilk the lactase solution according to claim 3, and decomposing lactosecontained in the raw material milk at 1 to 60° C.
 19. A method fortreating a raw material milk, comprising adding to a raw material milkthe lactase solution according to claim 4, and decomposing lactosecontained in the raw material milk at 1 to 60° C.
 20. The method forproducing the lactase solution according to claim 9, wherein thesalting-out treatment is carried out at pH 4 to 9.